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Yrb2p Is a Nuclear Protein That Interacts with Prp20p, a Yeast Rcc1 Homologue

1997; Elsevier BV; Volume: 272; Issue: 50 Linguagem: Inglês

10.1074/jbc.272.50.31877

ISSN

1083-351X

Autores

Tetsuya Taura, Gabriel Schlenstedt, Pamela A. Silver,

Tópico(s)

RNA and protein synthesis mechanisms

Resumo

A conserved family of Ran binding proteins (RBPs) has been defined by their ability to bind to the Ran GTPase and the presence of a common region of approximately 100 amino acids (the Ran binding domain). The yeast Saccharomyces cerevisiae genome predicts only three proteins with canonical Ran binding domains. Mutation of one of these, YRB1, results in defects in transport of macromolecules across the nuclear envelope (Schlenstedt, G., Wong, D. H., Koepp, D. M., and Silver, P. A. (1995)EMBO J. 14, 5367–5378). The second one, encoded byYRB2, is a 327-amino acid protein with a Ran binding domain at its C terminus and an internal cluster of FXFG and FG repeats conserved in nucleoporins. Yrb2p is located inside the nucleus, and this localization relies on the N terminus. Results of both genetic and biochemical analyses show interactions of Yrb2p with the Ran nucleotide exchanger Prp20p/Rcc1. Yrb2p binding to Gsp1p (yeast Ran) as well as to a novel 150-kDa GTP-binding protein is also detected. The Ran binding domain of Yrb2p is essential for function and for its association with Prp20p and the GTP-binding proteins. Taken together, we suggest that Yrb2p may play a role in the Ran GTPase cycle distinct from nuclear transport. A conserved family of Ran binding proteins (RBPs) has been defined by their ability to bind to the Ran GTPase and the presence of a common region of approximately 100 amino acids (the Ran binding domain). The yeast Saccharomyces cerevisiae genome predicts only three proteins with canonical Ran binding domains. Mutation of one of these, YRB1, results in defects in transport of macromolecules across the nuclear envelope (Schlenstedt, G., Wong, D. H., Koepp, D. M., and Silver, P. A. (1995)EMBO J. 14, 5367–5378). The second one, encoded byYRB2, is a 327-amino acid protein with a Ran binding domain at its C terminus and an internal cluster of FXFG and FG repeats conserved in nucleoporins. Yrb2p is located inside the nucleus, and this localization relies on the N terminus. Results of both genetic and biochemical analyses show interactions of Yrb2p with the Ran nucleotide exchanger Prp20p/Rcc1. Yrb2p binding to Gsp1p (yeast Ran) as well as to a novel 150-kDa GTP-binding protein is also detected. The Ran binding domain of Yrb2p is essential for function and for its association with Prp20p and the GTP-binding proteins. Taken together, we suggest that Yrb2p may play a role in the Ran GTPase cycle distinct from nuclear transport. Movement of macromolecules into and out of the nucleus is a multistep process crucial for most cellular events, including transcriptional regulation, progression through the cell cycle, and DNA duplication. Studies with both higher eukaryotes and yeast have revealed much about the requirements for this highly conserved process (1Görlich D. Mattaj I.W. Science. 1996; 271: 1513-1518Crossref PubMed Scopus (1067) Google Scholar, 2Corbett A.H. Silver P.A. Microbiol. Mol. Biol. Rev. 1997; 61: 193-211Crossref PubMed Scopus (170) Google Scholar). Certain amino acid sequences (termed nuclear localization sequences (NLS) 1The abbreviations used are: NLS, nuclear localization sequence; NPC, nuclear pore complex; YRB2, yeast Ran binding protein 2; GAP, GTPase activating protein; PBS, phosphate-buffered saline; GMPPNP, 5′-guanylyl imidodiphosphate; RBP, Ran binding protein; 5-FOA, 5-fluoroorotic acid; PCR, polymerase chain reaction; kb, kilobase; HA, hemagglutinin; GST, glutathioneS-transferase; PIPES, 1,4-piperazinediethanesulfonic acid; GDPβS, guanosine 5′-O-2-(thio)diphospate.) are recognized by soluble transport factors in the cytoplasm and delivered to the nuclear pore complex (NPC) (3Pante N. 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Ponstingl H. Wittinghofer A. J. Biol. Chem. 1995; 270: 11860-11865Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 21Corbett 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, 22Melchior F. Weber K. Gerke V. Mol. Biol. Cell. 1993; 4: 569-581Crossref PubMed Scopus (78) Google Scholar), a nucleotide exchange factor termed Rcc1 (23Ohtsubo M. Kai R. Furuno N. Sekiguchi M. Hayashida H. Kuma K. Miyata S. Fukushige S. Murotsu T. Matsubara K. Nishimoto T. Genes Dev. 1987; 1: 585-593Crossref PubMed Scopus (156) Google Scholar) (Prp20p in yeast) (24Fleischmann M. Clark M.W. Forrester W. Wickens M. Nishimoto T. Aebi M. Mol. Gen. Genet. 1991; 227: 417-423Crossref PubMed Scopus (48) Google Scholar, 25Aebi M. Clark M.W. Vijayraghavan U. Abelson J. Mol. Gen. Genet. 1990; 224: 72-80Crossref PubMed Scopus (125) Google Scholar), and a protein termed Ntf2p (26Corbett A.H. Silver P.A. J. Biol. 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Saavedra C. Loeb J.D.J. Cole C.N. Silver P.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 225-229Crossref PubMed Scopus (168) Google Scholar). Additionally, yeast mutants in RNA1(encoding the Gsp1p/Ran GAP) are defective for bi-directional nuclear transport of proteins and RNAs (21Corbett 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, 29Hopper A.K. Banks F. Evangelidis V. Cell. 1978; 19: 211-219Abstract Full Text PDF Scopus (139) Google Scholar, 31Hutchinson H.T. Hartwell L.H. McLaughlin C.S. J. Bacteriol. 1969; 99: 807-814Crossref PubMed Google Scholar). The inability to regenerate the GTP-bound form of Ran either by mutation of Prp20p (13Koepp D.M. Wong D.H. Corbett A.H. Silver P.A. J. Cell Biol. 1996; 133: 1163-1176Crossref PubMed Scopus (114) Google Scholar,30Kadowaki T. Zhao Y. Tartakoff A.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2312-2316Crossref PubMed Scopus (91) Google Scholar, 32Amberg D.C. Goldstein A.L. Cole C.N. Genes Dev. 1992; 6: 1173-1189Crossref PubMed Scopus (306) Google Scholar) or overexpression of the GDP-bound form of Gsp1p in yeast (13Koepp D.M. Wong D.H. Corbett A.H. Silver P.A. J. Cell Biol. 1996; 133: 1163-1176Crossref PubMed Scopus (114) Google Scholar) also results in defects in nuclear transport of proteins and RNAs. However, Ran and its regulators have been implicated in a number of other nuclear processes. These include chromatin condensation (23Ohtsubo M. Kai R. Furuno N. Sekiguchi M. Hayashida H. Kuma K. Miyata S. Fukushige S. Murotsu T. Matsubara K. Nishimoto T. Genes Dev. 1987; 1: 585-593Crossref PubMed Scopus (156) Google Scholar, 34Sazer S. Nurse P. EMBO J. 1994; 13: 606-615Crossref PubMed Scopus (109) Google Scholar), RNA processing (25Aebi M. Clark M.W. Vijayraghavan U. Abelson J. Mol. Gen. Genet. 1990; 224: 72-80Crossref PubMed Scopus (125) Google Scholar, 35Hopper A.K. Traglia H.M. Dunst R.W. J. Cell Biol. 1990; 111: 309-321Crossref PubMed Scopus (167) Google Scholar, 36Kadowaki T. Goldfarb D. Spitz L.M. Tartakoff A.M. Ohno M. EMBO J. 1993; 12: 2929-2937Crossref PubMed Scopus (154) Google Scholar), cell cycle progression (37Ren M. Drivas G. D'Eustachio P. Rush M.G. J. Cell Biol. 1993; 120: 313-323Crossref PubMed Scopus (154) Google Scholar, 38Nishitani H. Ohtsubo M. Yamashita K. Iida H. Pines J. Yasudo H. Shibata Y. Hunter T. Nishimoto T. EMBO J. 1991; 10: 1555-1564Crossref PubMed Scopus (150) Google Scholar, 39Matsumoto T. Beach D. Cell. 1991; 66: 347-360Abstract Full Text PDF PubMed Scopus (179) Google Scholar), DNA replication (40Dasso M. Nishitani H. Kornbluth S. Nishimoto T. Newport J.W. Mol. Cell. Biol. 1992; 12: 3337-3345Crossref PubMed Scopus (72) Google Scholar, 41Kornbluth S. Dasso M. Newport J. J. Cell Biol. 1994; 125: 705-719Crossref PubMed Scopus (108) Google Scholar), and nuclear envelope assembly (25Aebi M. Clark M.W. Vijayraghavan U. Abelson J. Mol. Gen. Genet. 1990; 224: 72-80Crossref PubMed Scopus (125) Google Scholar, 42Demeter J. Morphew M. Sazer S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1436-1440Crossref PubMed Scopus (82) Google Scholar). It remains to be determined whether or not these phenotypes are simply a secondary consequence of defects in nuclear transport or reflect processes other than nuclear transport that are directly controlled by Ran. A conserved protein family has recently been defined by their ability to bind to Ran. These so-called ran bindingproteins (RBPs) have in common a stretch of about 100 amino acids that is necessary for Ran binding (43Beddow 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). Mammalian RanBP1 (18Coutavas E. Ren M. Oppenheim J.D. D'Eustachio P. Rush M.G. Nature. 1993; 366: 585-587Crossref PubMed Scopus (226) Google Scholar, 44Bischoff F.R. Krebber H. Smirnova E. Dong W. Ponstingl H. EMBO J. 1995; 14: 705-715Crossref PubMed Scopus (331) Google Scholar,45Lounsbury K.M. Beddow A.L. Macara I.G. J. Biol. Chem. 1994; 269: 11285-11290Abstract Full Text PDF PubMed Google Scholar) and its yeast homologue, Yrb1p (46Schlenstedt G. Wong D.H. Koepp D.M. Silver P.A. EMBO J. 1995; 14: 5367-5378Crossref PubMed Scopus (136) Google Scholar, 47Ouspenski 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), stimulate Ran GTPase by further activating the GAP activity (44Bischoff F.R. Krebber H. Smirnova E. Dong W. Ponstingl H. EMBO J. 1995; 14: 705-715Crossref PubMed Scopus (331) Google Scholar, 46Schlenstedt G. Wong D.H. Koepp D.M. Silver P.A. EMBO J. 1995; 14: 5367-5378Crossref PubMed Scopus (136) Google Scholar). Both proteins bind stably to the GTP-bound but not the GDP-bound form of Ran (18Coutavas E. Ren M. Oppenheim J.D. D'Eustachio P. Rush M.G. Nature. 1993; 366: 585-587Crossref PubMed Scopus (226) Google Scholar, 44Bischoff F.R. Krebber H. Smirnova E. Dong W. Ponstingl H. EMBO J. 1995; 14: 705-715Crossref PubMed Scopus (331) Google Scholar, 45Lounsbury K.M. Beddow A.L. Macara I.G. J. Biol. Chem. 1994; 269: 11285-11290Abstract Full Text PDF PubMed Google Scholar, 46Schlenstedt G. Wong D.H. Koepp D.M. Silver P.A. EMBO J. 1995; 14: 5367-5378Crossref PubMed Scopus (136) Google Scholar). In yeast, YRB1 is essential for cell growth, and temperature-sensitive mutants display nuclear transport defects (46Schlenstedt G. Wong D.H. Koepp D.M. Silver P.A. EMBO J. 1995; 14: 5367-5378Crossref PubMed Scopus (136) Google Scholar,47Ouspenski 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), consistent with the role as a Ran-regulator. Some portion of Yrb1p is located at the nuclear envelope and may provide a “docking” site for Ran at the nuclear pore complex (46Schlenstedt G. Wong D.H. Koepp D.M. Silver P.A. EMBO J. 1995; 14: 5367-5378Crossref PubMed Scopus (136) Google Scholar). However, Yrb1p was also identified in a screen for mutants of yeast with destabilized chromosomes (47Ouspenski 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), thus supporting the notion that there may be additional functions for Gsp1p and its regulators. Only two additional yeast proteins, Yrb2p and Nup2p, contain predicted Ran binding domains (48Hartmann E. Görlich D. Trends Cell Biol. 1995; 5: 192-193Abstract Full Text PDF PubMed Google Scholar, 49Dingwall C. Kandels-Lewis S. Séraphin B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7525-7529Crossref PubMed Scopus (74) Google Scholar). Like the mammalian RanBP2/Nup358 (50Wu J. Matunis M.J. Kraemer D. Blobel G. Coutavas E. J. Biol. Chem. 1995; 270: 14209-14213Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar,51Yokoyama N. Hayashi N. Seki T. Pante N. Ohba T. Nishii K. Kuma K. Hayashida T. Miyata U. Aebi U. Fukui M. Nishimoto T. Nature. 1995; 376: 184-188Crossref PubMed Scopus (413) Google Scholar), Nup2p is located at the yeast nuclear pore complex (52Loeb J.D.J. Davis L.I. Fink G.R. Mol. Biol. Cell. 1993; 4: 209-222Crossref PubMed Scopus (90) Google Scholar). Interestingly, Nup2p is dispensable for normal yeast cell growth (52Loeb J.D.J. Davis L.I. Fink G.R. Mol. Biol. Cell. 1993; 4: 209-222Crossref PubMed Scopus (90) Google Scholar). One possibility is that Nup2p and Yrb1p play redundant roles as Ran-docking sites. Yrb2p was identified as an open reading frame encoding a protein with similarity to the other Ran binding proteins (46Schlenstedt G. Wong D.H. Koepp D.M. Silver P.A. EMBO J. 1995; 14: 5367-5378Crossref PubMed Scopus (136) Google Scholar). The YRB2 coding sequence predicts a 36-kDa protein of 327 amino acids in length with the Ran binding domain at the C terminus. In addition, Yrb2p is predicted to contain two FXFG and three FG amino acid repeats that are typically found in nucleoporins. At its N terminus, Yrb2p is rich in charged and hydrophilic amino acids with several potential phosphorylation sites and short sequences similar to NLSs. Unlike Yrb1p, cells missing Yrb2p are viable except at low temperatures and show no obvious defects in nuclear transport (53Noguchi E. Hayashi N. Nakashima N. Nishimoto T. Mol. Cell. Biol. 1997; 17: 2235-2246Crossref PubMed Scopus (57) Google Scholar). Because of its similarity to Yrb1p and the important role of Ran-regulators in nuclear function, we now present results concerning the function of Yrb2p that appear to distinguish it from Yrb1p. Yrb2p is primarily localized in the nucleus. In addition, Yrb2p interacts and functions with Prp20p, based on both biochemical and genetic experiments. Yeast S. cerevisiaestrains used in this work are listed in Table I. Media for cell growth and genetic manipulations were according to standard procedures (54Guthrie C. Fink G.R. Methods in Enzymology, Guide to Yeast Genetics and Molecular Biology. 194. Academic Press, Inc., San Diego, CA1991Google Scholar, 55Rose M.D. Winston F. Hieter P. Methods in Yeast Genetics: A Laboratory Course Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1990Google Scholar). 5-Fluoroorotic acid (5-FOA) was added at 1 mg/ml if needed. Growth of cells was measured by counting the cell number directly or measurement of absorbance at 600 nm.Table IStrains used in this studyNameGenotypeSourcePSY362MATa npl3–1 ade2–1 his leu2–3,112 ura3–1Laboratory stockPSY712MATa prp20–101 his3 Δ200 leu2Δ1 ura3–52Laboratory stockPSY713MATα prp20–1 leu2Δ1 trp1Δ63 ura3–52Ref. 13Koepp D.M. Wong D.H. Corbett A.H. Silver P.A. J. Cell Biol. 1996; 133: 1163-1176Crossref PubMed Scopus (114) Google ScholarPSY714MATa leu2Δ1 trp1 ura3–52 rna1–1Ref. 21Corbett 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 ScholarPSY878MATa/α ade2/ade2 his3/his3 leu2/ leu2 trp1/trp1 ura3/ura3Ref. 13Koepp D.M. Wong D.H. Corbett A.H. Silver P.A. J. Cell Biol. 1996; 133: 1163-1176Crossref PubMed Scopus (114) Google ScholarPSY1002MATα Δyrb2::HIS3 ade2–1 his3–11,15 leu2–3,112 ura3–1 trp1This studyPSY1003MATa Δyrb2::HIS3 ade2–1 his3–11,15 leu2–3,112 ura3–1 trp1This studyPSY1004MATα YRB2 ade2–1 his3–11,15 leu2–3,112 ura3–1 trp1This studyPSY1005MATa YRB2 ade2–1 his3–11,15 leu2–3,112 ura3–1 trp1This studyFY23MATa leu2Δ1 trp1Δ63 ura3–52F. Winston (Harvard Medical School)FY86MATα his3Δ200 leu2Δ1 ura3–52F. WinstonJLY381MATα nup2–5::HIS3 trp1Δ63 ura3–52 leu2–3,112 his3Δ200 ade2G. Fink (Massachusetts Institute of Technology)L4596MATα nup1–2::LEU2 his3Δ200 leu2–3,112 ura3–52/pNUP1(CEN URA3)G. FinkLG230MATα rat7–1 his3Δ200 leu2Δ1 ura3–52Ref. 64Gorsch L.C. Dockendorff T.C. Cole C.N. J. Cell Biol. 1995; 129: 939-955Crossref PubMed Scopus (166) Google Scholar Open table in a new tab All DNA manipulations were according to standard procedures (56Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Janssen K. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1987Google Scholar, 57Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Positions of oligonucleotides used for polymerase chain reaction (PCR) are described by numbers when A of the initiation codon is positioned as +1. The YRB2 gene was cloned by inserting two PCR fragments generated with yeast genomic DNA as a template into pBluescriptKS(+) (Stratagene). Primers used for PCR were: GS59 (−654 to −636 with a KpnI site, 5′-CTGCCGATCTGGTACCACC-3′), GS64 (an antisense primer, 109 to 88, 5′-CCCTCGGTCTCTTAGGAGTTCC-3′), GS61 (1 to 20 with a BamHI site, 5′-CGGGATCCATGAGTGAGACCAATGGTGG-3′), and GS60 (an antisense primer, 1080 to 1062 with a XbaI site, 5′-GCTCTAGACGGGGTCCAGGGTGCGACC-3′). After generating 0.75- and 1.08-kb PCR fragments with the combinations of GS59/GS64 and GS61/GS60, respectively, the DNA was treated with KpnI andPstI (for GS59/GS64) or PstI and XbaI (GS61/GS60), and both fragments were inserted together intoKpnI-XbaI sites of pBluescriptKS(+) to yield pPS842. For YRB2 expression in yeast cells, pPS844 and pPS848 were constructed as follows: a KpnI-XbaI 1.73-kb fragment prepared from pPS842 or aBamHI-XbaI double digested 1.08-kb PCR fragment prepared with GS61/GS60 and genomic DNA as template (see above) were inserted into KpnI-XbaI sites of a CEN URA3 vector, pRS316 (58; for pPS844 construction), orBamHI-XbaI sites of a 2μ URA3 pGALvector, pPS293 (59; for pPS848). Influenza hemagglutinin (HA) epitope-tagged YRB2(YRB2::HA) plasmids were prepared by placing the tag between the 6th and 7th codons of YRB2 using the two additional primers TT7 (5′-CCGCTCGAGTACCCATACGACGTCCCAGACTACGCTGGCAATGCAGCCAGGG-3′), which carries a XhoI site and the HA coding sequence followed by 19 to 34 nucleotides of YRB2 coding sequence, and TT8 (5′-CCGCTCGAGACCATTGGTCTCACTCATGC-3′), a primer carrying aXhoI site followed by an anti-coding sequence of 18 to −2. pPS842 was used as a template for PCR with GS59/TT8 and TT7/GS60 to produce 0.67- and 1.10-kb fragments, respectively. After digestion withKpnI and AvaI (for GS59-TT8 fragment) orAvaI and XbaI (for TT7-GS60), the fragments were inserted into the KpnI-XbaI sites of pRS316 to yield pPS1081. YRB2::HA fragment amplified from pPS1081 with TT9 (5′-CGGGATCCATGAGTGAGACCAATGG-3′), a primer with a BamHI site and the first 17 bases of theYRB2 coding region, and GS60 was cloned into pPS293 usingBamHI and XbaI sites to generate pPS1082. A 1.08-kb YRB2 fragment generated for the construction of pPS848 was placed on the 3′ end of GST (encoding glutathioneS-transferase gene) of pPS892 (13Koepp D.M. Wong D.H. Corbett A.H. Silver P.A. J. Cell Biol. 1996; 133: 1163-1176Crossref PubMed Scopus (114) Google Scholar) by usingBamHI-XbaI sites to yield pPS1083. 471 with aEcoRI site (5′-CGGAATTCAACATACTGAAAAAC-3′), TT14; anti-coding of 588–572 with a SmaI site (5′-TCCCCCGGGTTTTGGTTTGTCTTTTG-3′), TT15; 982–1000 with aSmaI site (5′-TCCCCCGGGTAAGTAAAGAGAAATAACG-3′). After treatment of the PCR fragments with proper restriction enzymes (AvaI and EcoRI for the TT7-TT21 fragment,EcoRI and XbaI for TT17-GS60, AvaI for the TT7-TT14, AvaI and XbaI for TT15-GS60), fragments were ligated into XhoI-XbaI digested pPS1081 to replace YRB2 with the truncated genes. For construction of a YRB2 chimeric gene with the Yrb1p Ran binding domain, we produced two PCR fragments. A 0.50-kb fragment, carrying HA followed codons 7 to 157 of YRB2, was generated with primers TT7 and TT18 (anti-coding of 471 to 451 with an EcoRI site, 5′-CGGAATTCGTTTTTCAGTATGTTGAAGCC-3′) and with pPS842 as a template. A 0.53-kb PCR fragment was obtained with TT19 (anEcoRI primer which codes codons 30 to 34 of YRB1, 5′-CGGAATTCGGTGGTAAGAAGGCCG-3′), TT20 (a SmaI primer carrying anti-coding sequence of codons 194 to 201 of YRB1, 5′-CCCCCGGGAGCCTTTTTGTTGATTTCTTGAG-3′), and pPS849 (46Schlenstedt G. Wong D.H. Koepp D.M. Silver P.A. EMBO J. 1995; 14: 5367-5378Crossref PubMed Scopus (136) Google Scholar) as a template. After digestion of both fragments with AvaI andEcoRI, we replaced the YRB2 part of pPS1086 (ΔC) with these fragments by using XhoI-SmaI sites that were treated with AvaI to yield pPS1087. Allyrb2 deletion mutants and the chimeric gene were recloned into pPS293 to make these genes galactose inducible (GAL-ΔN, pPS1088; GAL-ΔNup, pPS1089;GAL-ΔC, pPS1090; GAL-Chimera, pPS1091) and into pPS892 to produce GST-tagged versions (GST-ΔN, pPS1092; GST-ΔNup, pPS1093; GST-ΔC, pPS1094; GST-Chimera, pPS1095). All PCR reactions for plasmid constructions were carried out with Pfu DNA polymerase (Stratagene) or Vent DNA polymerase (New England Biolabs). YRB2was deleted as described by Baudin et al. (60Baudin A. Ozier-Kalogeropoulos O. Denouel A. Lacroute F. Cullin C. Nucleic Acids Res. 1993; 21: 3329-3330Crossref PubMed Scopus (1127) Google Scholar) with the following modifications. A 1.10-kb DNA fragment, which carries theHIS3 gene as well as 46 bases flanking regions of theYRB2 open reading frame, was generated by PCR. The resulting fragment was introduced into diploid cells (PSY878; Ref. 13Koepp D.M. Wong D.H. Corbett A.H. Silver P.A. J. Cell Biol. 1996; 133: 1163-1176Crossref PubMed Scopus (114) Google Scholar), andHIS + transformants were selected. Colonies carrying HIS3 integrations at YRB2 were screened by PCR with GS59 and a HIS3 internal primer (5′-GCCTCATCCAAAGGCGC-3′). The integration was then confirmed by Southern blotting. Yeast cells were grown in complete media or selective media containing 2% (w/v) glucose to a density of 1–5 × 107 cells/ml. For expression of genes under control of the GAL promoter, cells were grown in selective media containing 2% raffinose to a density of 0.5–2 × 107 cells/ml, then galactose was added to 2% (w/v), and induction was conducted for 2–4 h. Cells were fixed by treatment with 1/10 volume of 37% formaldehyde for 60–90 min and prepared for immunofluorescence as described previously (46Schlenstedt G. Wong D.H. Koepp D.M. Silver P.A. EMBO J. 1995; 14: 5367-5378Crossref PubMed Scopus (136) Google Scholar). Mouse monoclonal antibody 12CA5 was used at 1/4000 dilution in 5% bovine serum albumin with 0.2% Tween 20 in PBS. Fluorescein isothiocyanate-conjugated secondary antibodies (Jackson Immunoresearch Laboratories) were used at 1/1000 dilution. Poly (A)+ mRNA was localized using a protocol from Amberg et al. (32Amberg D.C. Goldstein A.L. Cole C.N. Genes Dev. 1992; 6: 1173-1189Crossref PubMed Scopus (306) Google Scholar) with some modifications (59Lee M.S. Henry M. Silver P.A. Genes Dev. 1996; 10: 1233-1246Crossref PubMed Scopus (258) Google Scholar). Cells containing plasmids expressing GST protein fusions were induced by addition of 2% galactose for 2–4 h. Procedures for purification were based on our previous report (46Schlenstedt G. Wong D.H. Koepp D.M. Silver P.A. EMBO J. 1995; 14: 5367-5378Crossref PubMed Scopus (136) Google Scholar) with modifications and carried out at 4 °C if not otherwise mentioned. After harvesting, cells were washed twice with Buffer A (20 mm PIPES-KOH, pH 6.8, 0.25 msorbitol, 150 mm KOAc, 5 mmMg(OAc)2), frozen with liquid nitrogen, and stored at −80 °C before use. Cells were resuspended into PBSMT + PI (PBS, 3 mm KCl, 2.5 mm MgCl2, 0.5% Triton X-100, and each 5 μg/ml pepstatin A, leupeptin, antipain, and chymostatin), mixed with glass beads (about half the volume of cell suspension), and disrupted. After removal of cell debris by centrifugation, protein concentration of the lysate was determined with protein assay kit (Bio-Rad). The resulting cell lysate was mixed with glutathione-conjugated agarose beads (Glutathione-Sepharose 4B, 50% slurry, Pharmacia Biotech Inc.) and incubated for 1–2 h. Nonhydrolyzable GTP analogs (5′-guanylyl imidodiphosphate, GMPPNP, Sigma) or GDP analogs (guanosine 5′-O-2-(thio)diphospate, GDPβS, Sigma) were added to the lysate at 1 mm prior to the mixing with glutathione beads if necessary. Beads were washed twice with PBSMT+PI and twice with PBSM (PBS, 3 mm KCl, 2.5 mm MgCl2). Proteins bound to the beads were eluted by boiling for 5 min in SDS sample buffer (61; bound fraction). Supernatants were mixed with an equal volume of 2 × SDS sample buffer and boiled for 5 min (unbound fraction). Binding assays with Ntf2p-agarose beads (62Wong D.H. Corbett A.H. Kent H.M. Stewart M. Silver P.A. Mol. Cell. Biol. 1997; 17: 3755-3767Crossref PubMed Scopus (90) Google Scholar) were carried out essentially by the same procedure as for GST-tagged proteins. Cell lysates with or without 1 mm nonhydrolyzable GTP analogs were mixed with Ntf2p-agarose and incubated for 1 h. After washing the beads, both bound and unbound fractions were prepared as described above. Proteins were separated with 10 or 12.5% SDS-polyacrylamide gel electrophoresis (61Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar) and stained with silver nitrate (56Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Janssen K. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1987Google Scholar).

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