Portal de Periódicos da CAPES

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

2000; Elsevier BV; Volume: 275; Issue: 6 Linguagem: Inglês

10.1074/jbc.275.6.4081

1083-351X

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

Genomics and Chromatin Dynamics

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-sensitiveyrb1 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-β 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. 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-sensitiveyrb1 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-β 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. GTPase-activating protein Ran-binding protein-1 Ran-binding domain 5′-guanylylimidodiphosphate polymerase chain reaction isopropylthio-β-d-galactoside phenylmethylsulfonyl fluoride glutathione S-transferase polyacrylamide gel electrophoresis 4-morpholinepropanesulfonic acid nuclear export signal In eukaryotic cells, DNA replication and transcription are compartmentalized in the nucleus. Access to the nucleoplasm is provided by thousands of pores that penetrate the double-membrane envelope of the nucleus. These pores are complex structures that, while permitting the diffusion of small molecules, only allow the passage of most proteins and nucleic acids when they are associated with soluble factors called karyopherins, which are specialized for either import (importins) or export (exportins) (for reviews, see Refs. 1.Ohno M. Fornerod M. Mattaj I.W. Cell. 1998; 92: 327-336Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar, 2.Görlich D. EMBO J. 1998; 17: 2721-2727Crossref PubMed Scopus (286) Google Scholar, 3.Mattaj I.W. Englmeier L. Annu. Rev. Biochem. 1998; 67: 265-306Crossref PubMed Scopus (1004) Google Scholar, 4.Wozniak R.W. Rout M.P. Aitchison J.D. Trends Cell Biol. 1998; 8: 184-188Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 5.Talcott B. Moore M.S. Trends Cell Biol. 1999; 9: 312-318Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). The direction of transport and accumulation against a concentration gradient are driven by the Ran GTPase (6.Richards S.A. Carey K.L. Macara I.G. Science. 1997; 276: 1842-1844Crossref PubMed Scopus (40) Google Scholar, 7.Izaurralde E. Kutay U. Vonkobbe C. Mattaj I.W. Görlich D. EMBO J. 1997; 16: 6535-6547Crossref PubMed Scopus (493) Google Scholar), which cycles between GTP- and GDP-bound states, like other G proteins. Ran itself is predominantly nuclear, and likewise, the guanine nucleotide exchange factor for Ran, RCC1, is associated with chromatin, ensuring that nuclear Ran is largely GTP-bound (8.Seki T. Hayashi N. Nishimoto T. J. Biochem. (Tokyo). 1996; 120: 207-214Crossref PubMed Scopus (59) Google Scholar, 9.Renault L. Nassar N. Vetter I. Becker J. Klebe C. Roth M. Wittinghofer A. Nature. 1998; 392: 97-101Crossref PubMed Scopus (243) Google Scholar, 10.Ohtsubo M. Okazaki H. Nishimoto T. J. Cell Biol. 1989; 109: 1389-1397Crossref PubMed Scopus (286) Google Scholar). Nuclear Ran·GTP binds to importins and thereby dissociates incoming importin-cargo complexes; conversely, nuclear Ran·GTP cooperatively promotes formation of exportin-cargo complexes (11.Görlich D. Pante N. Kutay U. Aebi U. Bischoff F.R. EMBO J. 1996; 15: 5584-5594Crossref PubMed Scopus (528) Google Scholar, 12.Rexach M. Blobel G. Cell. 1995; 83: 683-692Abstract Full Text PDF PubMed Scopus (663) Google Scholar, 13.Fornerod M. Ohno M. Yoshida M. Mattaj I.W. Cell. 1997; 90: 1051-1060Abstract Full Text Full Text PDF PubMed Scopus (1734) Google Scholar, 14.Stade K. Ford C.S. Guthrie C. Weis K. Cell. 1997; 90: 1041-1050Abstract Full Text Full Text PDF PubMed Scopus (929) Google Scholar, 15.Ossareh-Nazari B. Bachelerie F. Dargemont C. Science. 1997; 278: 141-144Crossref PubMed Scopus (620) Google Scholar, 16.Askjaer P. Jensen T.H. Nilsson J. Englmeier L. Kjems J. J. Biol. Chem. 1998; 273: 33414-33422Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 17.Fukuda M. Asano S. Nakamura T. Adachi M. Yoshida M. Yanagida M. Nishida E. Nature. 1997; 390: 308-311Crossref PubMed Scopus (1022) Google Scholar, 18.Kutay U. Bischoff F.R. Kostka S. Kraft R. Görlich D. Cell. 1997; 90: 1061-1071Abstract Full Text Full Text PDF PubMed Scopus (501) Google Scholar). Importin·Ran·GTP complexes recycle back to the cytosol (19.Hieda M. Tachibana T. Yokoya F. Kose S. Imamoto N. Yoneda Y. J. Cell Biol. 1999; 144: 645-655Crossref PubMed Scopus (49) Google Scholar). Thus, as the result of both import and export, Ran·GTP ends up in the cytosol in association with a transport factor and must be released to permit further rounds of transport. Release is driven by GTP hydrolysis. The GTPase-activating protein (RanGAP),1 responsible for catalyzing hydrolysis is present both in the cytosol and is attached to fibrils that extend from the cytoplasmic face of the nuclear pores (20.Hopper A.K. Traglia H.M. Dunst R.W. J. Cell Biol. 1990; 111: 309-321Crossref PubMed Scopus (167) Google Scholar, 21.Bischoff F.R. Klebe C. Kretschmer J. Wittinghofer A. Ponstingl H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2587-2591Crossref PubMed Scopus (415) Google Scholar, 22.Corbett A.H. Koepp D.M. Schlenstedt G. Lee M.S. Hopper A.K. Silver P.A. J. Cell Biol. 1995; 130: 1017-1026Crossref PubMed Scopus (152) Google Scholar). However, RanGAP alone only weakly stimulates hydrolysis of Ran·GTP bound to importins or exportins (11.Görlich D. Pante N. Kutay U. Aebi U. Bischoff F.R. EMBO J. 1996; 15: 5584-5594Crossref PubMed Scopus (528) Google Scholar, 23.Floer M. Blobel G. J. Biol. Chem. 1996; 271: 5313-5316Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). A cofactor is required to permit efficient RanGAP action and to dissociate transport factor complexes from nuclear pores. In mammalian cells, two proteins, RanBP1 (24.Coutavas E. Ren M. Oppenheim J.D. D'Eustachio P. Rush M.G. Nature. 1993; 366: 585-587Crossref PubMed Scopus (226) Google Scholar) and Nup358 (also called RanBP2) (25.Yokoyama N. Hayashi N. Seki T. Pante N. Ohba T. Nishii K. Kuma K. Hayashida T. Miyata T. Aebi U. et al.Nature. 1995; 376: 184-188Crossref PubMed Scopus (411) Google Scholar, 26.Wu J. Matunis M.J. Kraemer D. Blobel G. Coutavas E. J. Biol. Chem. 1995; 270: 14209-14213Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar), can perform this function. RanBP1 contains one copy and Nup358 contains four copies of a highly conserved, 135-residue domain that can bind Ran·GTP with high affinity, form a ternary complex with Ran·GTP and importins, and co-activate RanGAP (27.Beddow A.L. Richards S.A. Orem N.R. Macara I.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3328-3332Crossref PubMed Scopus (100) Google Scholar, 28.Lounsbury K.M. Richards S.A. Perlungher R.R. Macara I.G. J. Biol. Chem. 1996; 271: 2357-2360Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 29.Lounsbury K.M. Macara I.G. J. Biol. Chem. 1997; 272: 551-555Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 30.Floer M. Blobel G. Rexach M. J. Biol. Chem. 1997; 272: 19538-19546Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 31.Bischoff F.R. Görlich D. FEBS Lett. 1997; 419: 249-254Crossref PubMed Scopus (203) Google Scholar). This domain is referred to as the Ran-binding domain or RanBD. Nup358 is a giant nucleoporin that is a component of the fibrils that extend into the cytosol from the cytoplasmic face of the nuclear pore and associates with a form of RanGAP that is modified via attachment of a small ubiquitin-like polypeptide, SUMO-1 or Smt3 (32.Mahajan R. Delphin C. Guan T.L. Gerace L. Melchior F. Cell. 1997; 88: 97-107Abstract Full Text Full Text PDF PubMed Scopus (1002) Google Scholar, 33.Matunis M.J. Wu J.A. Blobel G. J. Cell Biol. 1998; 140: 499-509Crossref PubMed Scopus (377) Google Scholar). A related protein in the nematode, Caenorhabditis elegans, Ranup96, contains 2 RanBDs (27.Beddow A.L. Richards S.A. Orem N.R. Macara I.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3328-3332Crossref PubMed Scopus (100) Google Scholar). The crystal structure of the first RanBD (RanBD1) from Nup358, in a complex with Ran bound to a non-hydrolyzable GTP analog (GppNHp), has been solved (34.Vetter I.R. Nowak C. Nishimoto T. Kuhlmann J. Wittinghofer A. Nature. 1999; 398: 39-46Crossref PubMed Scopus (242) Google Scholar). RanBD1 possesses a β-barrel fold, similar to those of pleckstrin homology, phosphotyrosine binding, and Wiskott-Aldrich syndrome protein homology-1 domains. The N terminus of RanBD1 loops around Ran, and the C-terminal extension of Ran almost completely encircles RanBD1, in a mutual embrace. The Switch I effector loop in Ran makes contact with an invariant sequence, EWKERG, within the RanBD (residues 66–71 in mouse RanBP1). Other conserved regions in the RanBD, such as an -RXXMRRD- motif (residues 87–93 in mouse RanBP1), also make direct contact with Ran. In contrast to Nup358, RanBP1 is a small (25 kDa) cytosolic protein. The budding yeast (Saccharomyces cerevisiae) homolog of RanBP1, Yrb1, is essential for viability (35.Ouspenski I.I. Mueller U.W. Matynia A. Sazer S. Elledge S.J. Brinkley B.R. J. Biol. Chem. 1995; 270: 1975-1978Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), and two temperature-sensitive mutations,yrb1-1 ts (E146D/F151S) andyrb1-2 ts (L55P) (numbering according to equivalent positions in mouse RanBP1), display defects in both nuclear protein import and RNA export (36.Schlenstedt G. Wong D.H. Koepp D.M. Silver P.A. EMBO J. 1995; 14: 5367-5378Crossref PubMed Scopus (136) Google Scholar). Yrb1(E146D/F151S), but not Yrb1(L55P), still binds to activated (GTP-bound) yeast Ran, Gsp1(G21V), at the restrictive temperature (36.Schlenstedt G. Wong D.H. Koepp D.M. Silver P.A. EMBO J. 1995; 14: 5367-5378Crossref PubMed Scopus (136) Google Scholar). Correspondingly, in vivo, the yrb1-1 ts mutant shows a less pronounced phenotype than theyrb1-2 ts mutant with respect to nuclear protein import; interestingly, however, the potency of these same mutations in preventing RNA export is the reverse (36.Schlenstedt G. Wong D.H. Koepp D.M. Silver P.A. EMBO J. 1995; 14: 5367-5378Crossref PubMed Scopus (136) Google Scholar). Yeast contains no obvious counterpart of Nup358, but other smaller proteins with less well conserved RanBDs are present, including Yrb2 and Nup2. However, unlike a yrbΔ1 null mutant, yrb2Δ andnup2Δ cells are viable. Thus, Yrb1 is most likely the primary agent available to bind to Ran·GTP in transport receptor complexes in the cytosol and to assist yeast RanGAP (Rna1) to catalyze GTP hydrolysis and dissociation of Ran·GTP from these complexes. There is some evidence, however, that RanBP1 and its homologs may possess other functions, distinct from Ran binding (37.Turi T.G. Mueller U.W. Sazer S. Rose J.K. J. Biol. Chem. 1996; 271: 9166-9171Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 38.Ouspenski I.I. Exp. Cell Res. 1998; 244: 171-183Crossref PubMed Scopus (23) Google Scholar). As one approach to examine the essential physiological function(s) of RanBP1, we first randomly mutagenized the RanBD segment of mouse RanBP1, generating a library of mutants, many of which contain alterations of conserved residues in the RanBD. Second, the RanBP1 mutants were expressed in and purified from bacteria and analyzed for the known biochemical properties of RanBP1. Finally, to correlate the defects observed in vitro (if any) with function in vivo, we examined the ability of each mutant RanBP1 to rescue viability of a yeast strain containing a novel temperature-sensitive allele, yrb1-51 ts (A53D) (numbered according to the equivalent position in mouse RanBP1), which shows dramatic defects in both nuclear protein import and RNA export at restrictive temperature. 2M. Künzler, J. Trueheart, E. Hurt, and J. Thorner, submitted for publication. 2M. Künzler, J. Trueheart, E. Hurt, and J. Thorner, submitted for publication. 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. The resulting oligonucleotides were then used in a polymerase chain reaction (PCR) with the murine RanBP1 in pGEX-2T (Amersham Pharmacia Biotech) as the template and a 3′-pGEX primer, using VentTM DNA polymerase (Amersham Pharmacia Biotech), so as to create a fragment encoding the 3′-end of the RanBP1 coding sequence. This product was then used in a second round of PCR together with a 5′-pGEX primer to produce the full-length RanBP1 coding sequence. The resulting PCR products were cut with BamHI and EcoRI and then ligated into pGEX-2T to create the library. Ligation was performed in a 100-μl volume, using all the digested DNA product, so as to obtain a representative library. Three sets of spiked oligonucleotides, corresponding to residues 29–43, 66–79, and 90–101 of the RanBD of RanBP1, were used. Colonies from the libraries were isolated at random, and the plasmid DNAs were sequenced to determine the location and number of mutations present. The method of Zhou et al. (39.Zhou Y.H. Zhang X.P. Ebright R.H. Nucleic Acids Res. 1991; 19: 6052Crossref PubMed Scopus (204) Google Scholar) was used to introduce mutations within RanBP1 by limited template PCR using Taq DNA polymerase. Because the error rate of the Taq produces, on average, only one mutation per 600 base pairs in about 35% of the product, a Ran overlay assay was used to screen for mutations that reduced Ran binding. The PCR reaction contained 5′- and 3′-pGEX primers and wild-type RanBP1 in pGEX-2T as the template. The resulting product was cut with BamHI and EcoRI and religated into pGEX, as described above. After transformation into Escherichia coli strain DH5α, colonies were pooled to produce the library. The bacteria were then spread onto LB + ampicillin plates at a dilution so as to yield ∼1000 colonies per plate. Nitrocellulose filters soaked in 1 mm isopropylthio-β-d-galactoside (IPTG) were laid onto the plates for 3 h. The filters were lifted, and bacterial colonies adhering to the filter were lysed, first by exposure to chloroform vapor for 30 min and second by incubation overnight in 150 mm NaCl, 20 mm Tris-HCl, pH 8.0, 1 mm EDTA plus 100 μmphenylmethylsulfonyl fluoride (PMSF), 0.1 mg of lysozyme/ml, 0.1 unit of DNase/ml, 1.5% bovine serum albumin, 0.05% SDS, and 0.05% Tween 20. The filters were then probed by incubation with Ran loaded with [α-32P]GTP, as described previously (40.Lounsbury K.M. Beddow A.L. Macara I.G. J. Biol. Chem. 1994; 269: 11285-11290Abstract Full Text PDF PubMed Google Scholar). After exposure of the nitrocellulose to x-ray film to detect Ran-binding colonies, the film was compared with the agar plates, and colonies that did not give rise to positive spots in the overlay assay were picked for further examination. As a secondary screen, the colonies apparently deficient in Ran binding were grown in liquid culture in the presence of 1 mm IPTG for 3 h and then lysed, and the extracts were subjected to polyacrylamide gel electrophoresis (PAGE) in the presence of SDS. After transfer to nitrocellulose and a Ran overlay assay, the nitrocellulose was incubated with anti-GST antibodies to confirm that full-length GST-RanBP1 was being produced. Plasmids encoding full-length protein that exhibited defective Ran binding were sequenced to identify the location and number of the mutations present. To generate GST fusions to Ran, to RanGAP, to RCC1, and to the RanBP1 mutants of interest, DH5α transformed with the appropriate pGEX-2T (Amersham Pharmacia Biotech) construct was grown to A 600 nm = 1 and then induced with 1 mm IPTG for 4–16 h at 23 °C. The GST fusion proteins were purified from cell lysates using a glutathione-Sepharose matrix (Amersham Pharmacia Biotech) and concentrated to 1 mg of protein/ml using a Centricon-10 device (Aminco). Where necessary, the GST was removed by cleavage with purified thrombin (provided by Paula Tracy, University of Vermont). Importin-5 (RanBP5) was expressed as an N-terminally (His)6-tagged protein from plasmid pQE70 (provided by Dirk Görlich, University of Heidelberg). S-tagged importin-β (provided by S. Adam, Northwestern University) was induced in bacterial strain BL21(DE3) containing a pET-p97 construct as described previously (41.Palacios I. Hetzer M. Adam S.A. Mattaj I.W. EMBO J. 1997; 16: 6783-6792Crossref PubMed Google Scholar) and purified by binding to S-agarose. Protein concentrations were determined by the Bradford protein assay or from their calculated extinction coefficients at 280 nm. Protein integrity during preparation was monitored by SDS-PAGE and staining with Coomassie Blue dye. Ran overlay assays were performed as described previously, using Ran loaded with [γ-32P]GTP (3000 Ci/mmol) (40.Lounsbury K.M. Beddow A.L. Macara I.G. J. Biol. Chem. 1994; 269: 11285-11290Abstract Full Text PDF PubMed Google Scholar). Solution binding assays were performed using 20 pmol of GST-RanBP1 (or mutants thereof) attached to glutathione-Sepharose beads (20 μl of a 50:50 buffer:beads slurry). Ran that had been preloaded with [γ-32P]GTP (10 μm; 5 Ci/mmol) was incubated with the beads for 30 min on ice and then rapidly washed, and the amount of radioactivity retained was counted in a liquid scintillation counter. Inhibition of RCC1-mediated GTP/GDP exchange on Ran was determined in a similar manner to that described previously, using recombinant RCC1 with [γ-32P]GTP-loaded Ran (42.Lounsbury K.M. Richards S.A. Carey K.L. Macara I.G. J. Biol. Chem. 1996; 271: 32834-32841Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Briefly, 80 pmol of Ran (produced by thrombin cleavage of GST-Ran) was loaded with 20 μCi of [α-32P]GTP (3000 Ci/mmol, NEN Life Science Products) and then diluted 10-fold in buffer containing 50 mm MOPS, pH 7.1, 1 mm dithiothreitol, 10 mmMgCl2, and 0.1 mg of bovine serum albumin/ml. RCC1 was expressed as a GST fusion protein and cleaved from the GST using thrombin. The [α-32P]GTP:Ran was diluted into reaction buffer (25 mm MOPS, pH 7.1, 6.25 mmMgCl2, and 0.63 mm each of GDP, GTP, and NaH2 PO4) plus GST-RanBP1 (or mutants) at the desired concentration. RCC1 was added at time 0 to a final concentration of 1.5 nm, in a volume of 50 μl, at 30 °C. At 3 min, 20 μl of the sample was subjected to filter binding through nitrocellulose and quantitated by scintillation counting. GTP dissociation rate constants (k off) were calculated assuming simple exponential decay of a single species. Curves were fit to the RanBP1 inhibition data assuming a competitive inhibition model, using Kaleidagraph software. Co-activation of RanGAP by RanBP1 was measured using recombinant GST-mouse RanGAP (42.Lounsbury K.M. Richards S.A. Carey K.L. Macara I.G. J. Biol. Chem. 1996; 271: 32834-32841Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Ran was loaded with [γ-32P]GTP, as described above. The Ran·[γ-32P]GTP complex was diluted into reaction buffer, plus the desired concentration of GST-RanBP1, and hydrolysis was initiated by addition of RanGAP to a final concentration of 2.5 nm, in 50 μl at 30 °C. Samples (20 μl) were removed after 3 min and filtered, as above. GTP hydrolysis rate constants (k cat) were determined assuming a single exponential, as above. Curves were fit to the data assuming that binding increases the rate constant for hydrolysis without altering the K m for RanGAP. To determine the ability of the RanBP1 mutants to facilitate RanGAP activity toward Ran·GTP in complex with an importin, importin-5 was used. N-terminally (His)6-tagged importin-5 was expressed in bacteria and purified using Ni2+-saturated nitrilotriacetic-agarose beads (43.Jakel S. Görlich D. EMBO J. 1998; 17: 4491-4502Crossref PubMed Scopus (421) Google Scholar). GAP assays were performed essentially as described by Deane et al. (44.Deane R. Schafer W. Zimmermann H.P. Mueller L. Görlich D. Prehn S. Ponstingl H. Bischoff F.R. Mol. Cell. Biol. 1997; 17: 5087-5096Crossref PubMed Google Scholar), using 0.6 nm Ran that had been loaded with [γ-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 [γ-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 mmdithiothreitol, 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.Lounsbury K.M. Richards S.A. Perlungher R.R. Macara I.G. J. Biol. Chem. 1996; 271: 2357-2360Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). After incubation at 4 °C for 90 min, complexes were captured onto glutathione-Sepharose beads (40 μl) and washed three times with binding buffer containing 0.1 mm GTP. Proteins were separated by SDS-PAGE. After transfer to nitrocellulose, bound proteins were detected using anti-Ran (Signal Transduction R32620/L1), anti-RanBP1 (Santa Cruz sc-1159), and anti-His (Qiagen anti-(His)5) antibodies. Binding to importin-β was performed using S-tagged importin-β attached to S-agarose beads (45.Chi N.C. Adam S.A. Mol. Biol. Cell. 1997; 8: 945-956Crossref PubMed Scopus (65) Google Scholar). Formation of a GST-RanBP1·Ran·GDP·importin-β complex was performed in a similar manner, except that [α-32P]GDP-loaded Ran replaced Ran(G19V), and the complex was detected by washing the beads and counting for bound radioactivity. Two vectors were used for heterologous expression of RanBP1 in yeast. To obtain relatively high level expression, the pYES2 vector (Invitrogen) was modified by introduction of a triple HA1 tag between the uniqueEcoRI/HindIII sites downstream of the galactose-inducible GAL1 promoter. The tag was designed to possess BamHI and EcoRI cloning sites at its 3′-end, in the same reading frame as in pGEX-2T, so as to facilitate subcloning between the two vectors. The resulting vector was called pYESH3. To create a low copy version of this vector, the segment (SmaI-ClaI) containing the 2-μm DNA origin of replication was deleted and replaced with a 1.5-kilobase pair fragment (SpeI-ClaI) containing CEN4-ARS that was excised from yCplac33 and had its 5′-overhang filled in by incubation with the Klenow fragment of E. coli DNA polymerase I and all four dNTPs, yielding pYECH3. Wild-type and mutant RanBP1 coding sequences were transferred from pGEX into both pYESH3 and pYECH3. To express HA-tagged RanBP1 mutants at levels comparable with the endogenous Yrb1, the 570-base pair promoter region 5′ to theYRB1 gene was amplified from yeast genomic DNA by PCR and subcloned (as a PstI-XbaI fragment) into yCplac22, yielding yCplacTY. The RanBP1 mutants of interest were then inserted into this vector, and the resulting plasmids were then introduced by DNA-mediated transformation into either yeast strain JY525 (MATa fus1Δ1 ura3-52 his4-Δ29 trp1-Δ63 ade2-101) or its otherwise isogenic derivative, JY604 (MATa yrb1-51 ts fus1Δ1 ura3-52 his4-Δ29 trp1-Δ63 ade2-101). Due to theyrb1-51 ts mutation strain JY604 cannot grow at a temperature above 30 °C, whereas the parental strain JY525 grows well, even at temperatures as high as 37 °C. Growth of JY604 at restrictive temperature is fully restored by expression of either YRB1 or mouse RanBP1 cDNA (HTF9a). 3J. Trueheart and J. Thorner, unpublished observations. Transformants were selected on synthetic medium containing 2% glucose but lacking uracil (SCGlc-Ura) at 20 °C. The resulting colonies were grown in liquid SCGlc-Ura medium under the same conditions and then patched onto agar plates containing the same medium with either glucose (2%) or galactose plus raffinose (2% each) as the carbon source and incubated either at 20 or 37 °C. To detect expression of the HA-tagged RanBP1 mutants in yeast, JY604 transformants were grown overnight in 5 ml of SCGlc-Ura at 23 °C. When the cultures reached an A 600 nm = 1.0, 3 ml of each was centrifuged, and the cell pellet was resuspended in 5 ml of SCGal/Raf-Ura and incubated at 30 °C for 4 h. The yeast were then harvested by centrifugation, washed with water, resuspended in 0.1 ml of water containing protease inhibitors (4 μmaprotinin, 30 μm leupeptin, and 250 μmPMSF), and disrupted by addition of boiling SDS-PAGE sample buffer followed by vigorous vortex mixing with glass beads. 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). Libraries of RanBP1 mutants containing mutations within the RanBD sequence were constructed in two ways (see under “Experimental Procedures”). For the libraries of mutants produced using spiked oligonucleotides, no selection or screen was performed because mutations were introduced at a defined rate and within a known region of the RanBD. Rather, clones were chosen at random and sequenced. Mutant sequences were obtained at a frequency of about 15%. To identify mutants from the library obtained using template-limited PCR, we developed and applied an overlay method for screening colonies to assess the ability of the RanBP1 mutants to associate with Ran·GTP (Fig. 1). Bacteria containing pGEX alone or mutated pGEX-RanBP1 were plated onto LB agar plus ampicillin and grown overnight. To induce expression of GST or the GST fusion proteins, the colonies were overlaid with a nitrocellulose filter soaked in IPTG and incubated for 3 h. Colonies adherent to the filter were lysed, and the released filter-bound proteins were incubated with [α-32P]GTP:Ran and then washed to remove unbound probe. Colonies expressing GST alone did not retain detectable radioactivity, upon exposure to x-ray film. In contrast, colonies expressing GST-RanBP1 bound [α-32P]GTP:Ran, and the exposed film could be readily aligned with the original colonies. Moreover, it was relatively easy to identify coloni