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J Biol Chem, Vol. 275, Issue 6, 4081-4091, February 11, 2000

Random Mutagenesis and Functional Analysis of the Ran-binding Protein, RanBP1^*

Clark Petersen, Nicholas Orem^§, Joshua Trueheart^¶, Jeremy W. Thorner^¶, and Ian G. Macara^**

From the  Center for Cell Signaling, University of Virginia, Charlottesville, Virginia 22908, ^§ Dartmouth College, Hanover, New Hampshire 03755, and the ^¶ Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3202

Ran GTPase is required for nucleocytoplasmic transport of many types of cargo. Several proteins that recognize Ran in its GTP-bound state (Ran·GTP) possess a conserved Ran-binding domain (RanBD). Ran-binding protein-1 (RanBP1) has a single RanBD and is required for RanGAP-mediated GTP hydrolysis and release of Ran from nuclear transport receptors (karyopherins). In budding yeast (Saccharomyces cerevisiae), RanBP1 is encoded by the essential YRB1 gene; expression of mouse RanBP1 cDNA rescues the lethality of Yrb1-deficient cells. We generated libraries of mouse RanBP1 mutants and examined 11 mutants in vitro and for their ability to complement a temperature-sensitive yrb1 mutant (yrb1-51^ts) in vivo. In 9 of the mutants, the alteration was a change in a residue (or 2 residues) that is conserved in all known RanBDs. However, 4 of these 9 mutants displayed biochemical properties indistinguishable from that of wild-type RanBP1. These mutants bound to Ran·GTP, stimulated RanGAP, inhibited the exchange activity of RCC1, and rescued growth of the yrb1-51^ts yeast cells. Two of the 9 mutants altered in residues thought to be essential for interaction with Ran were unable to rescue growth of the yrb1^ts mutant and did not bind detectably to Ran in vitro. However, one of these 2 mutants (and 2 others that were crippled in other RanBP1 functions) retained some ability to co-activate RanGAP. A truncated form of RanBP1 (lacking its nuclear export signal) was able to complement the yrb1^ts mutation. When driven from the YRB1 promoter, 4 of the 5 mutants most impaired for Ran binding were unable to rescue growth of the yrb1^ts cells; remarkably, these mutants could nevertheless form ternary complexes with importin-5 or importin- and Ran-GTP. The same mutants stimulated only inefficiently RanGAP-mediated GTP hydrolysis of the Ran·GTP·importin-5 complex. Thus, the essential biological activity of RanBP1 in budding yeast correlates not with Ran·GTP binding per se or with the ability to form ternary complexes with karyopherins, but with the capacity to potentiate RanGAP activity toward GTP-bound Ran in these complexes.

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