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

Clark Petersen,Nicholas Orem,Joshua Trueheart,Jeremy W Thorner,Ian G Macara

doi:10.1074/jbc.275.6.4081

Abstract

Ran GTPase is required for nucleocytoplasmic transport of many types of cargo. Several proteins that recognize Ran in its GTP-bound state (Ran x 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 x 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-beta and Ran-GTP. The same mutants stimulated only inefficiently RanGAP-mediated GTP hydrolysis of the Ran x GTP x importin-5 complex. Thus, the essential biological activity of RanBP1 in budding yeast correlates not with Ran x 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.

Highlights

In eukaryotic cells, DNA replication and transcription are compartmentalized in the nucleus
Assays of binding to importin-5 were performed in 1.0 ml of Ran binding buffer (20 mM MOPS, pH 7.1, 100 mM sodium acetate, 5 mM magnesium acetate, 5 mM dithiothreitol, 0.05% Tween), using 8 nM importin-5, 20 nM GST-Ran-binding protein-1 (RanBP1), plus wild-type Ran (20 nM) that had been preloaded with GTP [28]
To identify mutants from the library obtained using template-limited polymerase chain reaction (PCR), we developed and applied an overlay method for screening colonies to assess the ability of the RanBP1 mutants to associate with Ran1⁄7GTP (Fig. 1)

Summary

EXPERIMENTAL PROCEDURES

Mutant Library Construction Using Spiked Oligonucleotides—This method utilized oligonucleotides synthesized in such a fashion that each position (other than the first 6 and last 6 bases) was spiked at 6% with a mixture of all four phosphoramidates, to give an error rate of about 0.045. GAP assays were performed essentially as described by Deane et al [44], using 0.6 nM Ran that had been loaded with [␥-32P]GTP and preincubated for 30 min in the presence or absence of 40 nM GST RanBP1 and/or 40 nM importin-5. RanGAP was added to a final concentration of 400 nM, for 5 min at 30 °C, and [␥-32P]GTP remaining bound to the Ran was determined by filter binding, as described above. Assays of binding to importin-5 were performed in 1.0 ml of Ran binding buffer (20 mM MOPS, pH 7.1, 100 mM sodium acetate, 5 mM magnesium acetate, 5 mM dithiothreitol, 0.05% Tween), using 8 nM importin-5, 20 nM GST-RanBP1, plus wild-type Ran (20 nM) that had been preloaded with GTP [28]. Insoluble materials were removed by centrifugation, and the protein extracts were resolved by SDS-PAGE, transferred to nitrocellulose, and analyzed for expression of the (HA1)3-tagged RanBP1 proteins by immunoblotting with an anti-HA1 monoclonal antibody (12CA5)

RESULTS

DISCUSSION

Activates RanGAP?