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• W1981281290 abstract "The small Ras-like GTPase Ran plays an essential role in the transport of macromolecules in and out of the nucleus and has been implicated in spindle (1Gruss O.J. Carazo-Salas R.E. Schatz C.A. Guarguaglini G. Kast J. Wilm M. Le Bot N. Vernos I. Karsenti E. Mattaj I.W. Cell. 2001; 104: 83-93Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar, 2Nachury M.V. Maresca T.J. Salmon W.C. Waterman-Storer C.M. Heald R. Weis K. Cell. 2001; 104: 95-106Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar) and nuclear envelope formation (3Zhang C. Clarke P.R. Science. 2000; 288: 1429-1432Crossref PubMed Scopus (165) Google Scholar, 4Bamba C. Bobinnec Y. Fukuda M. Nishida E. Curr. Biol. 2002; 12: 503-507Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar) during mitosis in higher eukaryotes. We identified Saccharomyces cerevisiae open reading frame YGL164c encoding a novel RanGTP-binding protein, termed Yrb30p. The protein competes with yeast RanBP1 (Yrb1p) for binding to the GTP-bound form of yeast Ran (Gsp1p) and is, like Yrb1p, able to form trimeric complexes with RanGTP and some of the karyopherins. In contrast to Yrb1p, Yrb30p does not coactivate but inhibits RanGAP1(Rna1p)-mediated GTP hydrolysis on Ran, like the karyopherins. At steady state, Yrb30p localizes exclusively to the cytoplasm, but the presence of a functional nuclear export signal and the localization of truncated forms of Yrb30p suggest that the protein shuttles between nucleus and cytoplasm and is exported via two alternative pathways, dependent on the nuclear export receptor Xpo1p/Crm1p and on RanGTP binding. Whereas overproduction of the full-length protein and complete deletion of the open reading frame reveal no obvious phenotype, overproduction of C-terminally truncated forms of the protein inhibits yeast vegetative growth. Based on these results and the exclusive conservation of the protein in the fungal kingdom, we hypothesize that Yrb30p represents a novel modulator of the Ran GTPase switch related to fungal lifestyle. The small Ras-like GTPase Ran plays an essential role in the transport of macromolecules in and out of the nucleus and has been implicated in spindle (1Gruss O.J. Carazo-Salas R.E. Schatz C.A. Guarguaglini G. Kast J. Wilm M. Le Bot N. Vernos I. Karsenti E. Mattaj I.W. Cell. 2001; 104: 83-93Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar, 2Nachury M.V. Maresca T.J. Salmon W.C. Waterman-Storer C.M. Heald R. Weis K. Cell. 2001; 104: 95-106Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar) and nuclear envelope formation (3Zhang C. Clarke P.R. Science. 2000; 288: 1429-1432Crossref PubMed Scopus (165) Google Scholar, 4Bamba C. Bobinnec Y. Fukuda M. Nishida E. Curr. Biol. 2002; 12: 503-507Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar) during mitosis in higher eukaryotes. We identified Saccharomyces cerevisiae open reading frame YGL164c encoding a novel RanGTP-binding protein, termed Yrb30p. The protein competes with yeast RanBP1 (Yrb1p) for binding to the GTP-bound form of yeast Ran (Gsp1p) and is, like Yrb1p, able to form trimeric complexes with RanGTP and some of the karyopherins. In contrast to Yrb1p, Yrb30p does not coactivate but inhibits RanGAP1(Rna1p)-mediated GTP hydrolysis on Ran, like the karyopherins. At steady state, Yrb30p localizes exclusively to the cytoplasm, but the presence of a functional nuclear export signal and the localization of truncated forms of Yrb30p suggest that the protein shuttles between nucleus and cytoplasm and is exported via two alternative pathways, dependent on the nuclear export receptor Xpo1p/Crm1p and on RanGTP binding. Whereas overproduction of the full-length protein and complete deletion of the open reading frame reveal no obvious phenotype, overproduction of C-terminally truncated forms of the protein inhibits yeast vegetative growth. Based on these results and the exclusive conservation of the protein in the fungal kingdom, we hypothesize that Yrb30p represents a novel modulator of the Ran GTPase switch related to fungal lifestyle. GTPase-activating protein guanine-nucleotide exchange factor glutathione S-transferase nuclear export signal open reading frame protein A RanGTP-binding domain wild-type green fluorescent protein guanosine 5′-3-O-(thio)triphosphate tobacco etch virus protease cleavage site nuclear localization signal thrombin cleavage site GTPases of the Ras superfamily act as molecular switches in a number of cellular processes (5Takai Y. Sasaki T. Matozaki T. Physiol. Rev. 2001; 81: 153-208Crossref PubMed Scopus (2061) Google Scholar). The two states of the switch are the GDP- and the GTP-bound forms of the GTPase where the GTP-bound state is the “on”-state based on its interaction with downstream effectors or target proteins. These target proteins represent the molecular links between a given GTPase and the regulated cellular process. The Ras-like GTPase Ran (Gsp1p in Saccharomyces cerevisiae) is an abundant, soluble protein shuttling between the cytoplasm and the nucleoplasm with a predominant localization in the nucleoplasm at steady state. Typical for all members of this superfamily, Ran has low intrinsic GTP hydrolysis and guanine-nucleotide exchange activities, which are activated by a specific cytoplasmic GTPase-activating protein (GAP)1 (RanGAP1/Rna1p) and a nuclear guanine-nucleotide exchange factor (RanGEF) (RCC1/Prp20p), respectively. Besides RanGAP1 and RanGEF, several Ran-binding proteins have been identified (6Künzler M. Hurt E. J. Cell Sci. 2001; 114: 3233-3241PubMed Google Scholar). Binding of most of these proteins is restricted to either the GDP- or the GTP-bound state of the GTPase. The major classes of RanGTP-binding proteins are the RanBP1 homologous proteins, which act as coactivators of RanGAP1-mediated GTP hydrolysis on Ran, and the family of nuclear transport receptors or karyopherins. Binding of RanGTP to latter class of proteins is the basis of the essential role of Ran in nucleocytoplasmic transport (6Künzler M. Hurt E. J. Cell Sci. 2001; 114: 3233-3241PubMed Google Scholar, 7Komeili A. O'Shea E.K. Annu. Rev. Genet. 2001; 35: 341-364Crossref PubMed Scopus (54) Google Scholar, 8Macara I.G. Microbiol. Mol. Biol. Rev. 2001; 65: 570-594Crossref PubMed Scopus (740) Google Scholar). Ran has also been implicated in spindle and nuclear envelope formation in higher eukaryotes, and recent reports (1Gruss O.J. Carazo-Salas R.E. Schatz C.A. Guarguaglini G. Kast J. Wilm M. Le Bot N. Vernos I. Karsenti E. Mattaj I.W. Cell. 2001; 104: 83-93Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar, 2Nachury M.V. Maresca T.J. Salmon W.C. Waterman-Storer C.M. Heald R. Weis K. Cell. 2001; 104: 95-106Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar, 9Zhang C. Hutchins J.R. Muhlhausser P. Kutay U. Clarke P.R. Curr. Biol. 2002; 12: 498-502Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) suggest that karyopherins are also the target proteins in these two processes. Here we report the results of a two-hybrid screen with the aim to identify novel Ran-binding proteins in the yeast S. cerevisiae and the characterization of a yeast ORF, YGL164c, which was found to encode a novel RanGTP-binding protein. The yeast strains used in this work are listed in TableI. Chromosomal tagging ofYRB30 with ProtA or GFP was performed in the RS453 strain according to Longtine et al. (10Longtine M.S. McKenzie III, A. Demarini D.J. Shah N.G. Wach A. Brachat A. Philippsen P. Pringle J.R. Yeast. 1998; 14: 953-961Crossref PubMed Scopus (4169) Google Scholar) and confirmed by PCR and immunoblot analysis.Table IList of yeast strains usedYeast strainsCharacteristicsSourceL40Δgal4MATahis3-delta200 trp1–901 leu2–3,112 ade2 lys2–801 am gal4::KanMX6 LYS2::(lexAop)4-HIS3 URA3::(lexAop)8-lacZRef.38Fromont-Racine M. Mayes A.E. Brunet-Simon A. Rain J.-C. Colley A. Dix I. Decourty L. Joly N. Ricard F. Beggs J.D. Legrain P. Yeast. 2000; 17: 95-110Crossref PubMed Google ScholarY187MATα ura3–52 his3–200 ade2–101 trp1–901 leu2–3,112 met-gal4Δ gal80Δ URA3::GAL1UAS-GAL1TATA-lacZRef.38Fromont-Racine M. Mayes A.E. Brunet-Simon A. Rain J.-C. Colley A. Dix I. Decourty L. Joly N. Ricard F. Beggs J.D. Legrain P. Yeast. 2000; 17: 95-110Crossref PubMed Google ScholarW303–1AMATa ura3–1 trp1–1 his3–11,15 leu2–3,112 ade2–1 can1–100 GALR. S. FullerW303–1A rna1–1MATa ura3–1 trp1–1 his3–11,15 leu2–3,112 ade2–1 can1–100 GAL rna1–1M. KünzlerYAB20MATa ura3–1 trp1–1 his3–11,15 leu2–3,112 ade2–1 can1–100 GAL yrb30::HIS3This studyYAB21MATα ura3–1 trp1–1 his3–11,15 leu2–3,112 ade2–1 can1–100 GAL yrb30::HIS3This studyYAB22MATa/α ura3–1/ura3–1 trp1–1/trp1–1 his3–11,15/his3–11,15 leu2–3,112/leu2–3,112 ade2–1/ade2–1 can1–100/can1–100 GAL/GAL yrb30::HIS3/yrb30::HIS3This studyCRM1T539CMATa ura3–1 trp1–1 his3–11,15 leu2–3,112 ade2–1 can1–100 GAL xpo1::KAN pRS315-CRM1T539CRef. 13Neville M. Rosbash M. EMBO J. 1999; 18: 3746-3756Crossref PubMed Scopus (159) Google ScholarRS453MATa ura3–52 trp1–1 leu2–3 ade2–1 his3–11,15 can1–100Ref. 14Segref A. Sharma K. Doye V. Hellwig A. Huber J. Luhrmann R. Hurt E. EMBO J. 1997; 16: 3256-3271Crossref PubMed Scopus (435) Google ScholarRS453 YRB30-ProtAMATa ura3–52 trp1–1 leu2–3 ade2–1 his3–11,15 can1–100 YRB30-ProtA::TRP1This studyRS453 YRB30-GFP(S65T)MATa ura3–52 trp1–1 leu2–3 ade2–1 his3–11,15 can1–100 YRB30-GFP::HIS3This studyYAB15W303–1A msn5::TRP1This studyAJH54MATacse1::LEU2 ade2–101 trp1-delta901 ura3–52 leu2–3,112 his3–11,15 pRS314-cse1–2Ref. 39Schroeder A. Chen X. Xiao Z. Fitzgerald-Hayes M. Mol. Gen. Genet. 1999; 261: 788-795Crossref PubMed Scopus (12) Google ScholarY0546RS453los1::HIS3K. HellmuthY1717W303–1A xpo1::KAN pRS313-xpo1–1F. Stutz/M. KünzlerNMD3 shuffleMATa his3 leu2 lys2 ura3 TRP1 nmd3::KanMX4 (ARS/CEN URA3 NMD3)Ref. 33Gadal O. Strauss D. Kessl J. Trumpower B. Tollervey D. Hurt E. Mol. Cell. Biol. 2001; 21: 3405-3415Crossref PubMed Scopus (257) Google Scholar Open table in a new tab Transformation of yeast cells with DNA was performed using a modified version of the lithium acetate method (11Gietz D. St Jean A. Woods R.A. Schiestl R.H. Nucleic Acids Res. 1992; 20: 1425Crossref PubMed Scopus (2894) Google Scholar). Unless indicated otherwise, yeast cells were cultivated at 30 °C. Preparation of standard yeast media was described previously (12Kaiser C. Michaelis S. Mitchell A. Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1994Google Scholar). Induction with galactose was performed by adding an equal volume of YPGal to cells grown in selective medium containing 2% (w/v) raffinose as sole carbon source (SRC). Leptomycin B treatment of CRM1T539C cultures was done as described (13Neville M. Rosbash M. EMBO J. 1999; 18: 3746-3756Crossref PubMed Scopus (159) Google Scholar). Growth of yeast and Escherichia coli, plasmid recovery, mating, and tetrad analysis were done as described previously (14Segref A. Sharma K. Doye V. Hellwig A. Huber J. Luhrmann R. Hurt E. EMBO J. 1997; 16: 3256-3271Crossref PubMed Scopus (435) Google Scholar). Standard techniques were used for the manipulation of recombinant DNA (15Sambrook J. Russel D.W.E. Molecular Cloning, A Laboratory Manual, 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2001Google Scholar). PCR amplifications were performed using standard conditions (16Saiki R.K. Gelfand D.H. Stoffel S. Scharf S.J. Higuchi R. Horn G.T. Mullis K.B. Erlich H.A. Science. 1988; 239: 487-494Crossref PubMed Scopus (13491) Google Scholar) and Vent DNA polymerase (New England Biolabs, Beverly, MA). Fusions between wt and mutant GSP1and the DNA-binding domain of E. coli LexA protein were constructed by inserting PCR-generated StuI-PstIGSP1 fragments into the SmaI-PstI sites of the two-hybrid vector pBTM116 (17Bartel P.L. Fields S. Methods Enzymol. 1995; 254: 241-263Crossref PubMed Scopus (303) Google Scholar). For pNOPPATA1L-YRB30 and pGALPATG1L-YRB30 the whole ORF of YRB30 was amplified from genomic DNA as a NcoI-BamHI fragment and inserted into the corresponding sites of pNOPPATA1L (pUN100-NOP1p::ProtA-TEV::ADH1t) (17Bartel P.L. Fields S. Methods Enzymol. 1995; 254: 241-263Crossref PubMed Scopus (303) Google Scholar) or pGALPATG1L (pUN100- GAL1p::ProtA-TEV::GAL4t) (17Bartel P.L. Fields S. Methods Enzymol. 1995; 254: 241-263Crossref PubMed Scopus (303) Google Scholar), respectively. The GFP-YRB30 fusion gene was generated by subcloning the ORF of YRB30 as a 1.4-kb PstI fragment from pNOPPATA1L-YRB30 into pNOPGFP2L (pRS425-NOP1p::GFP(S65T)) (17Bartel P.L. Fields S. Methods Enzymol. 1995; 254: 241-263Crossref PubMed Scopus (303) Google Scholar). Recloning of theNOP1p::GFP(S65T)::YRB30::GAL4tfusion gene as a SacI-KpnI fragment from pNOPGFP2L-YRB30 into the corresponding sites of pRS424 or pRS426 yielded analogous plasmids with TRP1 orURA3 marker genes, respectively. pET9d-GST-TEV-YRB30 was generated by replacing aNcoI-SacI fragment containing the MAD2gene from pET9d-GST-TEV-MAD2 (G. Stier, European Molecular Biology Laboratory (EMBL) Heidelberg, Germany) with the corresponding fragment from pGALPATG1L-YRB30. For expression of a His6-tagged fusion protein in E. coli the entire YRB30 ORF was ligated as aNcoI-BamHI fragment (see above) into pET9d-His6-TB adjacent to the Thrombin cleavage site (G. Stier, EMBL). The various plasmids containing deleted or mutatedYRB30 genes were constructed in an analogous way. Fusions between the bipartite NLS of ribosomal protein L25 under control of its own promoter and GFP-YRB30 were constructed by inserting YRB30 as PstI fragment from pNOPGFP2L-YRB30 into pRS314-L25NLS-GFP-ARC1 2K. Galani and E. Hurt, unpublished data.between GFP and the coding sequence of ARC1. The various plasmids containing deleted or mutated YRB30 genes were constructed by PCR in an analogous way. For assessing the functionality of the C-terminal NES in theYRB30 ORF various PCR-generated BamHI fragments of YRB30 were introduced into the BclI site of pRS315-NMD3(ΔNES1+2)-eGFP, which will be described elsewhere. 3T. Gerstberger and E. Hurt, manuscript in preparation. To check the correct orientation of the insert a NcoI site was introduced immediately upstream of the 3′-BamHI site. For overexpression studies, YRB30 was expressed under control of the galactose-inducible GAL1-promoter from high copy number vector pRS426GAL1 (18Mumberg D. Müller R. Funk M. Nucleic Acids Res. 1994; 22: 5767-5768Crossref PubMed Scopus (803) Google Scholar). For this purpose, full-length and truncated YRB30 ORFs were cloned as PCR-generatedSpeI-XhoI fragments. Expression of all proteins was verified by Western blot analysis using the anti-Yrb30p antiserum or commercially available anti-ProtA, anti-GFP, anti-His6, or anti-GST antibodies. Plasmids pNOPPATA1L-GSP1wt and pNOPPATA1L-GSP1(G21V) were described previously (19Lau D. Künzler M. Braunwarth A. Hellmuth K. Podtelejnikov A. Mann M. Hurt E. J. Biol. Chem. 2000; 275: 467-471Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). pRS315-NMD3-eGFP and pRS315-NMD3(ΔNES1/2)-eGFP were kindly provided by T. Gerstberger. Plasmids for expression of GST, His6-Gsp1wt, His6-Yrb1, His6-Rna1, GST-Yrb1, His6-Xpo1, and GST-Kap95 were described previously (20Enenkel C. Blobel G. Rexach M. J. Biol. Chem. 1995; 270: 16499-16502Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 21Künzler M. Gerstberger T. Stutz F. Bischoff F.R. Hurt E. Mol. Cell. Biol. 2000; 20: 4295-4308Crossref PubMed Scopus (47) Google Scholar). E. coli expression plasmids for His6-Gsp1wt (T. Gerstberger) and His6-Gsp1ΔDE (this study) were derived from pTrcHisA-GSP1(G21V) (22Yan C. Lee L.H. Davis L.I. EMBO J. 1998; 17: 7416-7429Crossref PubMed Scopus (205) Google Scholar) by replacing the NheI-HindIII GSP1(G21V) fragment by analogous PCR-generated fragments coding for Gsp1wt and Gsp1p (1–212). A plasmid for expression of His6-Kap95p inE. coli was constructed by inserting the KAP95ORF as BamHI fragment from pGEX4T3-KAP95 (1–861) (20Enenkel C. Blobel G. Rexach M. J. Biol. Chem. 1995; 270: 16499-16502Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar) into the BamHI site of pPROEX1. The YRB30 gene was disrupted by replacing the entire open reading frame of YRB30 with a PCR-generated HIS3-cassette in the diploid strain W303-D. The disruption was verified by PCR, and haploidyrb30::HIS3 strains were obtained by sporulation and tetrad dissection of the heterozygous diploid strain. Mating of haploid disruption strains of opposite mating type led to homozygous diploid disruption strains. A rabbit polyclonal antiserum against Yrb30p was raised against His6-Yrb30p fusion protein expressed in BL21(DE3) cells (23Studier F.W. Rosenberg A.H. Dunn J.J. Dubendorff J.W. Methods Enzymol. 1990; 185: 60-89Crossref PubMed Scopus (6003) Google Scholar) and affinity purified over Talon beads (Clontech, Palo Alto, CA). Immunization of two rabbits was performed by a commercial antibody service (J. Pineda, Berlin, Germany). For detection of Yrb30p in immunoblots, the resulting antisera were used at a dilution of 1:4000. The two-hybrid screen was performed as described previously (24Fromont-Racine M. Rain J.C. Legrain P. Nat. Genet. 1997; 16: 277-282Crossref PubMed Scopus (711) Google Scholar) but using L40Δgal4 cells for transformation of the bait-plasmids. In vitrointeraction between recombinant proteins was assayed as described previously (25Künzler M. Hurt E.C. FEBS Lett. 1998; 433: 185-190Crossref PubMed Scopus (78) Google Scholar). For the pull-down assay from yeast extract a culture from wt strain W303–1A was grown at 30 °C to an A600 of 1.5. Cells were harvested by centrifugation and lysed using a Fritsch Pulverisette 6 (Fritsch GmbH, Idar-Oberstein, Germany). Endogenous glutathione in the supernatant was removed by gel filtration using G25-fine-Sepharose (Amersham Biosciences). Loading of extract with GTPγS was done according to loading of Gsp1p with GTP (25Künzler M. Hurt E.C. FEBS Lett. 1998; 433: 185-190Crossref PubMed Scopus (78) Google Scholar). Purified GST-Yrb30p (∼15 μg) was rebound to 50 μl of GSH-Sepharose in universal buffer (25Künzler M. Hurt E.C. FEBS Lett. 1998; 433: 185-190Crossref PubMed Scopus (78) Google Scholar) containing 5 mmβ-mercaptoethanol and one tablet of Complete EDTA-free protease inhibitor mix (Roche Diagnostics) per 50 ml, washed, and incubated with ∼800 μg of protein of the corresponding supernatant. After washing the bound proteins were eluted in sample buffer and analyzed with SDS-PAGE and Coomassie Blue staining, as well as mass spectrometry. Affinity purification of ProtA fusion proteins was performed essentially as described (26Senger B. Simos G. Bischoff F.R. Podtelejnikov A. Mann M. Hurt E. EMBO J. 1998; 17: 2196-2207Crossref PubMed Scopus (157) Google Scholar). Assays were described previously (27Deane 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). GST-Yrb30p or GST-Yrb1p as a control were incubated in universal buffer (25Künzler M. Hurt E.C. FEBS Lett. 1998; 433: 185-190Crossref PubMed Scopus (78) Google Scholar) with Gsp1pGTP. After washing, the complexes were incubated for 12 min at 4 °C with His6-Rna1p or buffer alone. The released proteins were collected. After washing the bound fraction was eluted by boiling with sample buffer. Human Ran loaded with GTP and Rna1p from Schizosaccharomyces pombe were kindly provided by R. Bischoff (Deutsches Krebsforschungszentrum, Heidelberg, Germany). Classical and confocal fluorescence microscopy of living cells expressing GFP(S65T) fusion proteins was done as described previously (28Hellmuth K. Lau D.M. Bischoff F.R. Künzler M. Hurt E. Simos G. Mol. Cell. Biol. 1998; 18: 6374-6386Crossref PubMed Scopus (203) Google Scholar). SDS-PAGE, immunoblot, and preparation of yeast cell extracts were performed according to standard protocols. To identify novel yeast Ran-binding proteins a two-hybrid screen was performed using both wt (40 million of interactions tested) and two dominant mutant forms (G21V and T26N; 29 million or 46 million of interactions tested, respectively) of yeast Ran, Gsp1p. These mutant forms are locked in the GTP-bound and the GDP-bound state, respectively (29Lounsbury 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). Among the preys identified, we pulled out a number of already known Ran-binding proteins, such as members of the karyopherin family, the yeast homolog of RanBP1 (Yrb1p), and Mog1p (Table II). In addition, we identified a number of candidate novel Ran-binding proteins (Yrbs), most of them in the screen with the wt form of Gsp1p. Comparably few preys were identified with the mutant forms of Gsp1p.Table IIList of identified two-hybrid preysBaitPreyNumber of clones (number of independent fusions)FunctionGsp1 wtKAP123 (YRB4)2-aAlready known Yrb.1Karyopherin (importin)Gsp1 wtKAP109 (CSE1)2-aAlready known Yrb.1Karyopherin (exportin)Gsp1 wtNUP42 (RIP1)2-bProteins that bind to yeast Ran probably via three-hybrid interactions involving karyopherins.29 (6)NucleoporinGsp1 wtNUP1592-bProteins that bind to yeast Ran probably via three-hybrid interactions involving karyopherins.6 (3)NucleoporinGsp1 wtSLZ12-cORFs selected for further analysis.19 (7)MeiosisGsp1 wtSHE1 (YBL031W)2-cORFs selected for further analysis.2 (1)UnknownGsp1 wtSEC31Cell polarityGsp1 wtFRS24 (1)Protein synthesisGsp1 wtYER139C2-cORFs selected for further analysis.1UnknownGsp1 wtYEL043W2-cORFs selected for further analysis.2 (1)UnknownGsp1 T26NMOG12-aAlready known Yrb.5 (3)Multicopy suppressor of gsp/ts mutationsGsp1 T26NYPL009C2-cORFs selected for further analysis.3 (1)UnknownGsp1 T26NYBR225W2-cORFs selected for further analysis.1UnknownGsp1 G21VYRB12-aAlready known Yrb.10 (3)RanBP1Gsp1 G21VYGL164C (YRB30)2-cORFs selected for further analysis.3 (1)Unknown2-a Already known Yrb.2-b Proteins that bind to yeast Ran probably via three-hybrid interactions involving karyopherins.2-c ORFs selected for further analysis. Open table in a new tab As a first approach to confirm a physical interaction between these candidate Ran-binding proteins and Gsp1p, we fused selected ORFs (see Table II) to a TEV-cleavable ProtA moiety and expressed the fusion proteins under control of the NOP1 or the GAL1 promoter. After affinity purification over IgG-Sepharose we probed the column loads and the TEV eluates with a specific antiserum against Gsp1p. In none of the cases we could detect an enrichment of Gsp1p in the eluate (data not shown). The fact that one of the ORFs, YGL164C (YRB30), was found as the only prey besides Yrb1p in the two-hybrid screen with the G21V mutant, suggested that the protein bound specifically to the GTP-bound form of Gsp1p, which may be underrepresented in our cell lysate because of RanGAP1(Rna1p)-mediated GTP hydrolysis. We hypothesized that a putative Yrb30p-Gsp1pGTP complex would be stabilized in therna1–1 mutant strain, in which GTP hydrolysis is inhibited. We therefore expressed and affinity-purified the ProtA-tagged Yrb30p driven from a galactose-inducible GAL1 promoter from both wild-type and rna1–1 mutant yeast cells. As predicted, we could enrich Gsp1p using ProtA-Yrb30p when purified from therna1–1 background (Fig. 1). In contrast, no Gsp1p could be copurified with ProtA-Yrb30p from the wt strain or with ProtA alone. A similar result was obtained if Yrb30p was expressed from the constitutive NOP1 promoter (data not shown). In an inverse approach, we coexpressed GFP-tagged Yrb30p with ProtA-tagged Gsp1p in its wt form or in its G21V mutant form, respectively, under control of the NOP1 promoter in wt cells. Proteins were purified over ProtA-Sepharose and eluted by TEV cleavage. The eluates were analyzed by immunoblotting using a GFP antiserum. In agreement with the two-hybrid data we detected GFP-Yrb30p coeluting with ProtA-Gsp1(G21V) but not with the wt form of Gsp1p (Fig.1). We conclude that Yrb30, initially found in a two-hybrid screen with Gsp1(G21V), interacts specifically with the GTP-bound form of Gsp1pin vivo. To test whether Yrb30p could bind to Gsp1p directly, we performed pull-down assays using recombinant proteins produced in E. coli. We immobilized GST-Yrb30p, as well as GSTp alone, as a negative control and GST-Yrb1p (yRanBP1) and GST-Kap95p (yImpβ) as positive controls on GSH-Sepharose beads and analyzed binding of His6-tagged Gsp1p, loaded either with GTP or GDP, to these beads. Like the already known RanGTP-binding proteins, Yrb30p showed clear binding of Gsp1pGTP but not of Gsp1pGDP in this assay, which indicates that these proteins interact directly and specifically (Fig. 2A). Moreover, we could demonstrate that Yrb30p binds human Ran with the same specificity (data not shown). As an alternative approach to test the specificity of the interaction between Yrb30p and Gsp1pGTP, we tested whether we could pull down endogenous Gsp1p from a yeast extract using an excess of immobilized GST-Yrb30p. To convert all GTPases in the yeast extract to their GTP-bound state, half of the extract was loaded with GTPγS whereas the other half was mock-treated (see “Experimental Procedures”). Indeed, Yrb30p was able to pull down Gsp1p that was verified by mass-spectrometry but only from the GTPγS-treated extract (Fig.2B), suggesting a high specificity and affinity for Gsp1pGTP. Having established the direct interaction between Yrb30p and Gsp1pGTP we were interested in delimiting the domain of Yrb30p required for binding to Gsp1pGTP. For this purpose we created various N- and C-terminal truncations of GST-Yrb30p and tested them for binding to His6-Gsp1pGTP as outlined above. As shown in Fig. 3A only the N-terminal 127 amino acids of Yrb30p were dispensable for RanGTP binding. Hence, the minimal RBD of Yrb30p comprises residues 128 to 440. This domain is much larger than the RBD of RanBP1 (∼130 residues) and does not show any sequence similarity to the RBDs of RanBP1 or importin β, suggesting a novel motif for RanGTP binding (Fig. 3B, RBD). A BLAST search with the YRB30 coding sequence reveals homologues of Yrb30p in S. pombe(GenBankTM accession numbers CAA90468.2 andCAA21820.3), Ashbya gossypii, 4F. Dietrich, T. Gaffney, and P. Philippsen, personal communication.Neurospora crassa (GenBankTM accession numberCAC04441.1), Candida albicans (Candida data base (genolist.pasteur.fr/ CandidaDB) number CA5261) (Fig.3B), and Candida glabrata (GenBankTMaccession number AAA35271.1). These proteins show sequence identities of 30 to 40% and similarities of 50 to 60% with highly conserved sequence blocks that are spread over the entire sequence and not restricted to the RanGTP-binding domain. It was shown previously (30Vetter I.R. Arndt A. Kutay U. Görlich D. Wittinghofer A. Cell. 1999; 97: 635-646Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar) that importin β and RanBP1 bind to different surfaces on RanGTP thus enabling the formation of ternary complexes. To get an idea about the epitope on RanGTP recognized by Yrb30p, we tested how His6-Kap95p (yeast importin β) and His6-Yrb1p (yRanBP1) would interfere with the in vitro interaction between GST-Yrb30p and His6-Gsp1pGTP. In a first experiment, GST-Yrb30p was bound to GSH-Sepharose beads and incubated with a mixture of His6-Gsp1pGTP and His6-Yrb1p. With increasing concentrations of His6-Yrb1p the amount of His6-Gsp1pGTP bound to GST-Yrb30p was decreasing suggesting that Yrb1p and Yrb30p compete for an overlapping binding site on Gsp1GTP (Fig. 4A). To test whether Yrb30p could build trimeric complexes, like Yrb1p, with karyopherins, we incubated GST-Yrb30p pre-bound GSH-Sepharose with His6-Kap95p and His6-Gsp1p either in its GTP- or GDP-bound form. As shown in Fig. 4B Yrb30p was able to build trimeric complexes with Kap95p and Gsp1pGTP. In contrast to Yrb1p, no trimeric complexes were observed with His6-Gsp1pGDP. In summary, the binding site for Yrb30p on RanGTP appears to overlap with the one for RanBP1 but is distinct from the one for importin β. All RanGTP-binding proteins known so far, including the karyopherins and the RanBP1-related proteins, inhibit RanGEF (RCC1)-mediated GTP-to-GDP exchange on Ran, presumably by competition with RCC1 for binding to RanGTP. Accordingly, GST-Yrb30p also had an inhibitory effect on RCC1-mediated guanine nucleotide exchange (Fig.5A). The dissociation constant of the Yrb30p-Gsp1pGTP complex derived from the concentration of GST-Yrb30p necessary for half-maximal inhibition was 10 nm, which is ∼five times higher than the value for Yrb1p (21Künzler M. Gerstberger T. Stutz F. Bischoff F.R. Hurt E. Mol. Cell. Biol. 2000; 20: 4295-4308Crossref PubMed Scopus (47) Google Scholar) but still significantly lower than for importin β, suggesting a rather high affinity. Because Yrb30p showed similar binding characteristics like Yrb1p, the coactivator of RanGAP-mediated GTP hydrolysis on Ran, we checked whether Yrb30p would show a similar biochemical behavior. RanGAP-mediated GTP hydrolysis on RanGTP was measured as release of32P from Ran loaded with [γ-32P]GTP as described earlier (24Fromont-Racine M. Rain J.C. Legrain P. Nat. Genet. 1997; 16: 277-282Crossref PubMed Scopus (711) Google Scholar). Addition of GST-Yrb30p did not coactivate the reaction but acted as an inhibitor like karyopherins (Fig.5B). We were able to confirm this result in pull-down assays where we incubated pre-formed complexes between His6-Gsp1pGTP and GST-Yrb1p or GST-Yrb30p, respectively, with recombinant His6-Rna1p (Fig. 5C). Under the applied conditions, we observed release of Gsp1p only from Yrb1p but not from Yrb30p." @default.
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• W1981281290 title "Identification and Characterization of a Novel RanGTP-binding Protein in the Yeast Saccharomyces cerevisiae" @default.
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